Immune responses must be regulated to ensure effective pathogen elimination and self tolerance. ICOS and PD-L1 play key roles in regulating this critical balance between T cell activation and tolerance. During the current funding period, we have been investigating how ICOS and PD-L1 in regulate pathogenic vs. protective T cell responses. Our studies point to novel roles for both ICOS and PD-L1 In sustaining the function of regulatory T cell cells and maintaining T cell tolerance. We have found that ICOS does not control the induction of regulatory CD4 T cells during mucosal tolerance, but instead, appears necessary for sustaining CD4 regulatory cells during mucosal tolerance. ICOS, c-Maf and IL-21 coordinately act to promote differentiation of IL-10 producing regulatory type 1 (Tr1) cells, but ICOS appears to be crucial for maintaining IL-27 driven, IL-10 producing Tri cells. These findings lead us to hypothesize that ICOS, c-maf and lL-27 wori<in concert to regulate mucosal tolerance. Likewise, we have identified mechanisms by which PD-L1 controls tolerance: PD-L1 limits activation of self-reactive T cells, function of self-reactive effector cells and promotes induction and maintenance of adaptive regulatory T cells. PD-L1 on non-hematopoietic cells promotes tissue tolerance. PD-L1 and PD-1 have become new therapeutic targets in cancer and chronic infection, since blockade of PD-1 or PD-L1 can activate anti-viral or anti tumor immunity. In view of their key roles In regulating tolerance, further studies are needed to determine how to effectively minimize the risk of immune-mediated tissue damage and autoimmunity, while modulating PD-L1 and PD-1 to enhance virus or tumor control. Our discovery of the PD-L1 :B7-1 pathway leads us to ask whether PD-L1 :PD-1 and PD- L1:B7-1 interactions have unique or overiapping roles in T cell tolerance, and investigate the function of PD- L1 on T cells during tolerance. We hypothesize that PD-L1:B7-1, as well as PD-L1:PD-1 interactions, inhibit self-reactive T cell responses, and that PD-L1 has a T cell intrinsic role in controlling T cell responses. To test these hypotheses, our Specific Aims are to: 1) Investigate the inter-relationships among ICOS, IL-27 and c-maf during mucosal tolerance;2) Analyze the functional significance of the novel PD-L1:B7-1 pathway and relative contributions of PD-L1:B7-1 and PD-L1 interactions in regulating T cell tolerance and autoimmunity; 3) Investigate the role of PD-L 1 onT cells in regulating T cell tolerance;and 4) Dissect the roles of PD-L 1 on specific cell types in mucosal tolerance. These studies should complement each other to further our understanding of mechanisms that control tolerance and autoimmunity, and provide therapeutic insights. RELEVANCE (See instructions): These studies will provide new insights into how ICOS and PD-L1 regulate the balance between T cell activation and tolerance. The results of our studies will have implications for developing new therapies for human chronic viral infections, cancer, autoimmune diseases and increasing success of transplantation. Our findings may assist with approaches for controlling Treg plasticity and help determine how to tiest manipulate PD-L1 therapeutically to enhance pathogen or tumor control, and minimize autoimmunity/immunopathology.